The interaction of nanoparticles with biological systems is an important issue in biomedical and environmental sciences. In our proposed research we will exploit the selective laser desorption and ionization of ligands attached to nanomaterials to track cellular uptake and endosomal release of nanomaterials using mass spectrometry. In this approach the ligands serve as mass "barcodes", allowing the simultaneous tracking of multiple particles. In our proposed research we will take a multi-pronged approach to the systematic study of cellular uptake of nanoparticles that integrates analytical methodology development with synthesis and biology. Our research features three Aims that will be pursued in parallel: Aim 1: Rotello will fabricate nanoparticle with systematic variations in size and surface functionality. Aim 2: Rotello will incubate particles in multiplex fashion with different cell types, different lines of the same cell type, and isogenic variants of specific cell lines. Vachet will then use LDI to determine relative and absolute particle uptake. Once the fundamental parameters for cellular uptake have been established, Rotello and Vachet will use LDI-MS to quantify endosomal release. These studies will provide quantitative structure-activity correlations for cellular uptake and endosomal release of nanomaterials. Aim 3: Vachet will parametrically explore methods to optimize the sensitivity of LDI particle tracking and will investigate the analytical scope of this method. These results will provide enhanced probes for future applications of LDI for particle tracking in vivo. Our research will couple the nanoparticle synthesis and biological proficiency developed by the Rotello group with the instrumental and quantitative capabilities of Vachet. The goal of this R21 grant is two-fold: development and validation of multiplex analysis of cellular uptake of nanoparticles coupled with the application of this method to explore structure-activity properties of cellular uptake of nanomaterials. These results will be important in and of themselves in terms of delivery and diagnostic applications. Moreover, they will lay the foundation for the application of LDI for the tracking of nanoparticles in vivo and in the environment. PUBLIC HEALTH RELEVANCE: Tracking nanoparticles is an important issue in biomedical and environmental science. We will develop a strategy that allows simultaneous tracking of multiple types of nanoparticles and use this method to characterize cellular uptake of these materials.